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Clinical and Molecular Characterization of Patients with Fructose 1,6-Bisphosphatase Deficiency

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Clinical and Molecular Characterization of Patients with Fructose 1,6-Bisphosphatase Deficiency Article Clinical and Molecular Characterization of Patients with Fructose 1,6-Bisphosphatase Deficiency Niu Li 1,†, Guoying Chang 2,†, Yufei Xu 1, Yu Ding 2, Guoqiang Li 1, Tingting Yu 1, Yanrong Qing 1, Juan Li 2, Yiping Shen 1,3, Jian Wang 1,* and Xiumin Wang 2,* 1 Department of Medical Genetics and Molecular Diagnostic Laboratory, Shanghai Children’s Medical Center, Shanghai Jiaotong University School of Medicine, Shanghai 200127, China; [email protected] (N.L.); [email protected] (Y.X.); [email protected] (G.L.); [email protected] (T.Y.); [email protected] (Y.Q.); [email protected] (Y.S.) 2 Department of Endocrinology and Metabolism, Shanghai Children’s Medical Center, Shanghai Jiaotong University School of Medicine, Shanghai 200127, China; [email protected] (G.C.); [email protected] (Y.D.); [email protected] (J.L.) 3 Department of Laboratory Medicine, Boston Children’s Hospital, Boston, MA 02115, USA * Correspondence: [email protected] (J.W.); [email protected] (X.W.); Tel.: +86-21-3862-6161 (J.W. & X.W.); Fax: +86-21-5875-6923 (J.W. & X.W.) † These authors contributed equally to this work. Academic Editor: William Chi-shing Cho Received: 26 February 2017; Accepted: 17 April 2017; Published: 18 April 2017 Abstract: Fructose-1,6-bisphosphatase (FBPase) deficiency is a rare, autosomal recessive inherited disease caused by the mutation of the FBP1 gene, the incidence is estimated to be between 1/350,000 and 1/900,000. The symptoms of affected individuals are non-specific and are easily confused with other metabolic disorders. The present study describes the clinical features of four Chinese pediatric patients who presented with hypoglycemia, hyperlactacidemia, metabolic acidosis, and hyperuricemia. Targeted-next generation sequencing using the Agilent SureSelect XT Inherited Disease Panel was used to screen for causal variants in the genome, and the clinically-relevant variants were subsequently verified using Sanger sequencing. Here, DNA sequencing identified six variations of the FBP1 gene (NM_000507.3) in the four patients. In Case 1, we found a compound heterozygous mutations of c.704delC (p.Pro235GlnfsX42) (novel) and c.960_961insG (p.Ser321Valfs) (known pathogenic). In Case 2, we found a compound heterozygous mutations of c.825 + 1G>A and c.960_961insG (both were known pathogenically). In Case 3, a homozygous missense mutation of c.355G>A (p.Asp119Asn) (reported in ClinVar database without functional study) was found. Case 4 had a compound heterozygous mutations c.720_729del (p.Tyr241GlyfsX33) (novel) and c.490G>A (p.Gly164Ser) (known pathogenically). Further in vitro studies in the COS-7cell line demonstrated that the mutation of ASP119ASN had no impact on protein expression, but decreased the enzyme activity, and with which the clinical significance of Asp119Asn can be determined to be likely pathogenic. This report not only expands upon the known spectrum of variation of the FBP1 gene, but also deepens our understanding of the clinical features of FBPase deficiency. Keywords: fructose-1,6-bisphosphatase (FBPase) deficiency; targeted-next generation sequencing; FBP1 gene; functional study 1. Introduction Fructose-1,6-bisphosphatase (FBPase) is one of the key enzymes of gluconeogenesis that catalyzes the splitting of fructose-1,6-bisphosphate (FBP) into fructose 6-phosphate and inorganic phosphate [1]. The FBPase enzyme is mainly expressed in the liver and kidney, two distinct genes, Int. J. Mol. Sci. 2017, 18, 857; doi:10.3390/ijms18040857 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2017, 18, 857 2 of 13 FBP1 and FBP2, which encode human FBPase [2]. The FBP1 gene (OMIM#611570), which is mainly expressed in liver tissues, is located on 9q22.3 and consists of seven exons that encode 338 amino acids [3]. The FBP2 gene (OMIM#603027) was initially isolated from muscle tissues and encodes a 339–amino acid protein that shares a 77% amino acid sequence identity with the FBP1 protein [4](Figure S1, see Supplementary Material). Mutations in the FBP1 gene cause FBPase deficiency (OMIM#229700), which is characterized by an impairment of glucose synthesis from all gluconeogenic precursors. It is an autosomal recessive inherited disorder with clinical symptoms of hypoglycemia, ketosis, acidosis, hyperlactacidemia, convulsions, and coma [1]. The non-specific clinical features of FBPase deficiency are easily confused with several other metabolic disorders, such as glucose-6-phosphatase (G6P) deficiency and phosphoglucomutase 1 (PGM1) deficiency, especially in the absence of definitive biochemical findings [5,6]. Measurement of FBPase activity using liver biopsies or leukocytes is apparently the most reliable diagnostic approach to date, although its sensitivity remains relatively low and is generally considered as an invasive medical procedure [7,8]. In addition, the disorder may become fatal when patients develop severe hypoglycemia when glycogen reserves are insufficient (in newborns or fasting individuals) or after a fructose load [9]. Thus, the establishment of a rapid and accurate diagnostic method for FBPase deficiency is imperative. We hereby report the clinical features and the results of genetic testing of four patients strongly suspected of having inherited metabolic diseases. Using targeted next-generation sequencing (NGS) with the Agilent SureSelect XT Inherited Disease Panel (Agilent Technologies, Inc., Santa Clara, CA, USA), all of the patients were confirmed to harbor pathogenic variants in the FBP1 gene (NM_000507.3) and were diagnosed with FBPase deficiency. The functions of two variants (Asp119Asn and 704delC) were also evaluated by in vitro experimentation. 2. Results 2.1. Clinical Description Case 1 was a girl of five years and six months old. She was referred to the gastroenterology clinic of Shanghai Children’s Medical Center (SCMC) for the chief complaint of fever, vomiting, convulsions, and coma. She was the first child of healthy, non-consanguineous parents and was born at term via normal delivery. When she was 3 days old, she experienced vomiting and was diagnosed with hypoglycemia, hemorrhage of the digestive tract, and metabolic acidosis in a local hospital. Since she was two years old, the patient had been hospitalized several times in local hospitals for these conditions. However, her developmental milestones were normal. Additionally, her two-year- old brother exhibited similar syndromes when he stopped breastfeeding at five months. The laboratory findings of this first patient indicated hypoglycemia (glucose: 0.3 mmol/L), hyperlactacidemia (11.9 mmol/L), metabolic acidosis (pH: 6.97, base excess: −25.0 mmol/L), and hyperuricemia (uric acid: 875 μmol/L). Blood samples obtained during hypoglycemia showed insulin: 8.0 μIU/mL, adrenocorticotropic hormone (ACTH) > 1250 pg/mL, cortisol: 57.3 μg/dL. The electroencephalogram and ultrasound results were normal. The cranial magnetic resonance imaging (MRI) results showed the first patient to have a mild enlarged bilateral ventricle (Figure 1A,B). Ultrasound results of the abdomen, thyroid, and heart were normal. The patient was treated for her symptoms with an infusion of antibiotics, glucose, and bicarbonate, immediately. Case 2 was a girl of seven years and three months old and who was taken to the neurology ward at SCMC due to convulsions. The patient was a full-term baby and her parents were physical healthy, with a non-consanguineous marriage. Before she was born, the couple had undergone two induced abortions. Her neonatal period and infancy were normal. At four years old, she fell ill with vomiting and developed lactic acidosis with hypoglycemia and convulsions, for which she was brought to the emergency department. She had a recurrence of these symptoms at five years and 10 months. There was no positive family history. The laboratory results showed hypoglycemia (glucose: 0.6 mmol/L), hyperlactacidemia (12.5 mmol/L), metabolic acidosis (pH: 6.88, base excess: −23.1 mmol/L), and hyperuricemia (uric acid: 599 μmol/L). After glucose infusion, results of blood samples showed Int. J. Mol. Sci. 2017, 18, 857 3 of 13 insulin (23.8 μIU/mL), ACTH (9.13 pg/mL), and cortisol (8.9 μg/dL). The cranial MRI results showed her to have mildly enlarged left ventricle (Figure 1C,D). An ultrasound showed no abnormalities of the abdomen, thyroid, or heart. After treatment with an infusion of glucose to correct hypoglycemia, the patient was transferred to Department of Endocrinology for further therapy and investigation, where her dynamic blood was tested by glucose a continuous glucose monitoring system (CGMS, Medtronicn Minimed) for three days. The mean glucose level was 5.0 ± 0.8 mmol/L, the average area under the curve (AUC, <3.9 mmol/L) was 0.17, and hypoglycemia mostly occurred at night and in the morning (Figure 2A). Figure 1. Clinical features of the patients. (A,B) Magnetic resonance imaging (MRI) showed a mildly enlarged bilateral ventricle in Case 1; (C,D) A mildly enlarged left ventricle was found in Case 2 by MRI; (E,F) Several parts of the brain (cerebellum, frontoparietal, basal ganglia, and thalamus) in Case 4 had symmetrical patchy abnormal signals; (G) Magnetic resonance spectroscopy (MRS) revealed the brain of Case 4 had an abnormal increased lactate peak and reduced N-acetyl aspartate (NAA) peak, which is marked by the red and green arrows, respectively; (H) The facial picture of Case 4 with a strabismic right eye. Int. J. Mol. Sci. 2017, 18, 857 4 of 13 Figure 2. The dynamic blood glucose monitoring results. The blood glucose range was continuously detected for three days by an audiomonitor (Minimed Paradigm 722, Medtronic). (A) Hypoglycemia (<3.9 mmol/L) of Case 2 mainly occurred from 8:00 pm to 8:00 am; (B) Though no hypoglycemia was detected in Case 4, he did not appear to have a high postprandial blood glucose peak, which could be induced by oral glucose intake (the peak in 16:00–18:00). The dashed line represents the mean value of glucose over three days.
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